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JOURNAL CLUB
RECENTLY PUBLISHED
An in-depth look at fluorescent dyes
for organelle labeling
A review of reagents for fluorescence microscopy
of cellular compartments and structures, Parts I–III.
■■ “Part I: BacMam labeling and reagents for vesicular structures”
Dolman NJ, Kilgore JA, Davidson MW (2013) Curr Protoc Cytom
65:12.30.1–12.30.27.
■■ “Part II: Reagents for non-vesicular organelles” Kilgore JA, Dolman
NJ, Davidson MW (2013) Curr Protoc Cytom 66:12.31.1–12.31.24.
■■ “Part III: Reagents for actin, tubulin, cellular membranes, and whole
cell and cytoplasm” Kilgore JA, Dolman NJ, Davidson MW (2014) Curr
Protoc Cytom 67:12.32.1–12.32.17.
particular assay or environmental condition. Once a reagent is
chosen, validated protocols are not always available, optimizations are often required, and image examples can be hard
to locate. Moreover, when a problem arises during the course
of the experiments, identifying a solution can be difficult due
to a lack of troubleshooting notes in product manuals or
published references.
Choosing a fluorescent reagent for imaging cellular structures
A three-part series of review articles that help to clarify
and organelles in live or fixed cells can be daunting. Not only
these issues has recently been published in Current Protocols
must the excitation and emission spectra of the reagent match
in Cytometry. Authored by our R&D scientists Jason Kilgore and
your imaging instrument, but often you also need to choose from
Nick Dolman, who have developed and tested many Molecular
among a number of reagents—including organic dyes, drug
Probes® organelle stains, as well as imaging guru Michael
conjugates, and fluorescent protein constructs—that target the
Davidson of Florida State University, this series pulls together
same structure. Compatibility with the cells, with other probes
a wealth of references and personal knowledge that covers the
in the same experiment, and with the treatments and protocols
most common fluorescent reagents for nearly every cellular
you are using can lead to the question: How do I choose one
structure or organelle, focusing primarily on microscopic analysis.
fluorescent probe over another?
Each article is broken into sections by target structure or
Each fluorescent reagent comes with its own set of char-
organelle. The discussion includes a short history on the different
acteristics that may prove to be beneficial or detrimental for a
reagents that have been used and their characteristics. A featured
reagent is highlighted, along with a validated protocol for that
reagent with an estimate on protocol time, a troubleshooting
guide in table format, an image example, and relevant recipes
for stock and final solutions.
In Part I the authors focus on the BacMam reagents, which
have been used for transient transduction of fluorescent proteins targeted to specific cellular structures. They also discuss
options for vesicular organelles such as endosomes (featuring
pHrodo® dextran conjugates), lysosomes (featuring the organic
Figure 1. (Figure 12.30.4 from “Part I”) Peroxisome staining in HeLa
cells. HeLa cells were transduced with CellLight® Peroxisome-GFP to
transiently express a GFP fusion protein in peroxisomes. Cells were
counterstained with Hoechst® 33342 dye (blue, nuclei). Reprinted with
permission from Current Protocols in Cytometry.
dye LysoTracker® Red DND-99), peroxisomes (featuring the
BacMam reagent CellLight® Peroxisome-GFP, Figure 1), and
autophagosomes (featuring another BacMam reagent, the
Premo™ Autophagy Sensor GFP-LC3B).
© 2014 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.
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In Part II the authors discuss reagents for labeling the
In Part III the authors describe reagents for actin (featuring
endoplasmic reticulum and nuclear membranes (featuring the
Alexa Fluor® phalloidin conjugates, Figure 2), tubulin (featuring
ER-Tracker dyes), Golgi apparatus (featuring dye-labeled cer-
the drug conjugate Tubulin Tracker™ Green), cellular membranes
amides), mitochondria (featuring the organic dye MitoTracker®
(featuring plasma membrane labeling with fluorescent wheat
Red CMXRos, Figure 2), and nucleoli (featuring the nucleic acid
germ agglutinin (WGA) conjugates, Figure 3), and whole-cell
dye SYTO® RNASelect™ Green). The best nucleus-selective dyes
and cytoplasm reagents (featuring the organic dye CFSE, also
for live- and fixed-cell labeling are also highlighted.
called CFDA, SE).
™
All three articles in this Current Protocols in Cytometry
series—Part I, Part II, and Part III—are available through the
Wiley Online Library. ■
Selected products featured in this
Current Protocols in Cytometry series
Quantity
Cat. No.
Part I: BacMam labeling and reagents for vesicular structures
CellLight® Peroxisome-GFP, BacMam 2.0
1 mL
C10604
LysoTracker Red DND-99
20 x 50 µL
L7528
pHrodo® Red Dextran, 10,000 MW
0.5 mg
P10361
pHrodo Green Dextran, 10,000 MW
0.5 mg
®
®
P35368
Premo™ Autophagy Sensor LC3B-GFP, BacMam 2.0 1 kit
P36235
Premo™ Autophagy Sensor LC3B-RFP, BacMam 2.0 1 kit
P36236
Part II: Reagents for non-vesicular organelles
Figure 2. (Figure 12.31.6 from “Part II”) Mitochondria and actin staining in a BPAE cell. A live BPAE (bovine pulmonary artery endothelial)
cell was labeled with MitoTracker® Red CMXRos (red, mitochondria) and
then fixed, permeabilized, and labeled with Alexa Fluor® 488 phalloidin
(green, F-actin) and DAPI (blue, nuclei). Reprinted with permission from
Current Protocols in Cytometry.
7-Aminoactinomycin D (7-AAD)
1 mg
A1310
BODIPY FL C5-Ceramide, complexed to BSA
5 mg
B22650
BODIPY® TR Ceramide, complexed to BSA
5 mg
B34400
®
DAPI
10 mg
D1306
ER-Tracker™ Blue-White DPX
20 x 50 µL
E12353
ER-Tracker Green (BODIPY FL Glibenclamide)
100 µg
E34251
ER-Tracker™ Red (BODIPY® TR Glibenclamide)
100 µg
E34250
™
®
Hoechst 33342, Trihydrochloride, Trihydrate
100 mg
H1399
MitoTracker® Red CMXRos
20 x 50 µg
M7512
NBD C6-Ceramide, complexed to BSA
5 mg
N22651
SYTO® 9 Green-Fluorescent Nucleic Acid Stain
100 µL
S34854
®
SYTO 59 Red-Fluorescent Nucleic Acid Stain
100 µL
S11341
SYTO® RNASelect™ Green-Fluorescent Cell Stain
100 µL
S32703
®
Figure 3. (Figure 12.32.3 from “Part III”) Mitochondria and plasma
membrane staining in HeLa cells. Live HeLa cells were transfected
using pShooter™ vector pCMV/myc/mito/GFP and Lipofectamine® 2000
Transfection Reagent, stained with the Alexa Fluor® 594 conjugate
of wheat germ agglutinin (WGA) (red, plasma membrane), and then
imaged using confocal microscopy. Reprinted with permission from
Current Protocols in Cytometry.
36 | lifetechnologies.com
SYTOX® Green Nucleic Acid Stain
250 µL
S7020
TO-PRO®-3 Iodide (642/661)
1 mL
T3605
Part III: Reagents for actin, tubulin, cellular membranes,
and whole cell and cytoplasm
Alexa Fluor® 488 Phalloidin
300 units
A12379
5-(and 6-)Carboxyfluorescein Diacetate,
Succinimidyl Ester (CFSE; CFDA, SE)
25 mg
C1157
Tubulin Tracker™ Green (Oregon Green® Taxol,
Bis-Acetate)
1 set
T34075
Wheat Germ Agglutinin, Alexa Fluor® 594 Conjugate 5 mg
W11262
© 2014 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.